Denaturation enzymes

However, in most cases enzymes show lower activity in organic media than in water. This behavior has been ascribed to different causes such as diffusional limitations, high saturating substrate concentrations, restricted protein flexibility, low stabilization of the enzyme-substrate intermediate, partial enzyme denaturation by lyophilization that becomes irreversible in anhydrous organic media, and, last but not least, nonoptimal hydration of the biocatalyst [12d]. Numerous methods have been developed to activate enzymes for optimal use in organic media [13]. 

Enzymatic reactions are influenced by a variety of solution conditions that must be well controlled in HTS assays. Buffer components, pH, ionic strength, solvent polarity, viscosity, and temperature can all influence the initial velocity and the interactions of enzymes with substrate and inhibitor molecules. Space does not permit a comprehensive discussion of these factors, but a more detailed presentation can be found in the text by Copeland (2000). Here we simply make the recommendation that all of these solution conditions be optimized in the course of assay development. It is worth noting that there can be differences in optimal conditions for enzyme stability and enzyme activity. For example, the initial velocity may be greatest at 37°C and pH 5.0, but one may find that the enzyme denatures during the course of the assay time under these conditions. In situations like this one must experimentally determine the best compromise between reaction rate and protein stability. Again, a more detailed discussion of this issue, and methods for diagnosing enzyme denaturation during reaction can be found in Copeland (2000). 

When the rate of an enzyme catalyzed reaction is studied as a function of temperature, it is found that the rate passes through a maximum. The existence of an optimum temperature can be explained by considering the effect of temperature on the catalytic reaction itself and on the enzyme denaturation reaction. In the low temperature range (around room temperature) there is little denaturation, and increasing the temperature increases the rate of the catalytic reaction in the usual manner. As the temperature rises, deactivation arising from protein denaturation becomes more and more important, so the observed overall rate eventually will begin to fall off. At temperatures in excess of 50 to 60 °C, most enzymes are completely denatured, and the observed rates are essentially zero. 

Similarly to the above-mentioned entrapment of proteins by biomimetic routes, the sol-gel procedure is a useful method for the encapsulation of enzymes and other biological material due to the mild conditions required for the preparation of the silica networks [54,55]. The confinement of the enzyme in the pores of the silica matrix preserves its catalytic activity, since it prevents irreversible structural deformations in the biomolecule. The silica matrix may exert a protective effect against enzyme denaturation even under harsh conditions, as recently reported by Frenkel-Mullerad and Avnir [56] for physically trapped phosphatase enzymes within silica matrices (Figure 1.3). A wide number of organoalkoxy- and alkoxy-silanes have been employed for this purpose, as extensively reviewed by Gill and Ballesteros [57], and the resulting materials have been applied in the construction of optical and electrochemical biosensor devices. Optimization of the sol-gel process is required to prevent denaturation of encapsulated enzymes. Alcohol released during the... 

Monooxygenase Assays. Incubation media contained the following (final concentrations) 0.05M phosphate buffer, pH 7.A, glucose-6-phosphate (G-6-P, 2.3 mM), G-6-P dehydrogenase (3 units), NADP (0.23 mM), and KC1 (2.8 mM), and various tissue preparations. Substrates were added in small volumes (25 yl or less) of MeOH. Samples (1.1 ml) were shaken in a thermostated (usually at 22°C) water bath and reactions terminated by enzyme denaturation. Specific analytical procedures for aldrin epoxi-dation (13), 1 CH30-p-nitroanisole 0-demethylation (1A), and 3H-benzo(a)pyrene oxidation (15) have been described. 

Very recently evidence was provided that Hmd contains a low-molecular-mass, thermolabile cofactor that is tightly bound to the enzyme but could be released upon enzyme denaturation in urea or guanidinium chloride (Buurman et al. 2000). No indications were found that the cofactor contains a redox-active transition metal. Further studies are needed to determine the structure of the cofactor and its putative role in the catalytic mechanism. 

Biphasic systems consisting of ionic liquids and supercritical CO2 showed dramatic enhancement in the operational stability of both free and immobilized Candida antarctica lipase B (CALB) in the catalyzed kinetic resolution of rac- -phenylethanol with vinyl propionate at 10 MPa and temperatures between 120 and 150°C (Scheme 30) 275). Hydrophobic ionic liquids, [EMIM]Tf2N or [BMIM]Tf2N, were shown to be essential for the stability of the enzyme in the biotransformation. Notwithstanding the extreme conditions, both the free and isolated enzymes were able specifically to catalyze the synthesis of (J )-l-phenylethyl propionate. The maximum enantiomeric excess needed for satisfactory product purity (ee >99.9%) was maintained. The (S)-l-phenylethanol reactant was not esterified. The authors suggested that the ionic liquids provide protection against enzyme denaturation by CO2 and heat. When the free enzyme was used, [EMIM]Tf2N appeared to be the best ionic liquid to protect the enzyme, which... 

In both media water and w/o-microemulsions the enzymes denaturate with time. The complete immobilisation of the enzyme system in a continuous process leads to a limitation caused by the denaturation of the enzymes only. In order to obtain a complete immobilisation of the enzyme the transmembrane pressure of the ultrafiltration unit should often not exceed 1 bar [120]. 

Lyoprotectants can affect enzyme stability in both stages of lyophilization the freezing and the drying stages. In the freezing stage of lyophilization, ice crystals form and have been shown to be a cause of enzyme denaturation. Studies have shown that when added as a lyoprotectant, the amorphous polyol mannitol stabi-... 

Changes in the activity of enzymes may also occur by a variety of other parameters including temperature, pH, and ionic strength. Temperature can affect both the activity and the stability of the enzymes. For most enzymes, the reaction velocity doubles with a temperature increase of 10 C but potential enzyme denaturation may also occur (265). 

To prevent enzyme denaturation the vesicle preparation must be mild and simple. 

Effect of pH on enzyme denaturation Extremes of pH can also lead to denaturation of the enzyme, because the structure of the catalytically active protein molecule depends on the ionic charac ter of the amino acid side chains. 

Mix 1 ml of the enzyme preparation with the same volume of aniline solution in the PTFE cell immediately prior to electropolymerisation. Aniline containing acetylcholinesterase can be polymerised onto sonicated polydiaminobenzene-coated SPEs by sequentially cycling for 10 min between —200 and +800 mV versus Ag/AgCl at 50mV s-1. As the aniline polymerises at the exposed microelectrode elements, the polymer forms mushroom-like protrusions that extend outwards from the electrode surface and within which the acetylcholinesterase becomes entrapped. After polymerisation, the electrodes must be immediately submerged in pH 7.4 phosphate buffer at 4°C to prevent enzyme denaturation and stored at 4°C prior to use. 

Dr. Hamilton earlier observed that ether extraction of tissue induced autolysis, liberating active esterases and glycosi-dases, and thus leading to more free IAA than extraction of tissue by polar, and thus, enzyme-denaturing solvents (35). Thus, we knew then that there were enzymes in the tissue capaBTe of hydrolyzing IAA esters. Much later, Kopcewicz demonstrated the presence of an enzyme system which could synthesize lAA-myo-inositol from IAA, ATP, Mg++ and CoASH (43). More recently, Mr. Lech Michalczuk (unpublished) has shown that IAA-CoA will acylate inositol only in the presence of other nucleotides. Thus, the reaction is complex, but there is no doubt that enzymes to make and hydrolyze the IAA esters are present in corn. 

Alpha-amylase is most active at its pH optimum of 6.3 to 6.8.108,109 It is inactive at pH values below 4 and above 9. Enzymic starch conversion is terminated by raising the temperature until enzyme denaturation occurs or by the addition of enzyme poisons, such as the ions of copper, mercury or zinc. Inactivation can also be achieved by moving the pH outside the enzyme s active limits or by the addition of oxidizing agents, such as sodium hypochlorite, hydrogen peroxide or barium peroxide. 

Fig. 3.4 Temperature-dependent reconstitution of tetrameric K coli aspartase.29 A Reactivation of denatured aspartase. The enzyme denatured in 4 M guanidine-HCl was renatured at 4° C by dilution. After 14 min, the temperature of each preparation was shifted up as indicated in the figure. The temperature of each preparation was further shifted up to 30° C after 45 min. B HPLC analysis of intermediates in the renaturation process. Aspartase renatured at 4°C was incubated for 15 min at the indicated temperatures. An aliquot of each preparation was applied to a TSKgel G3000SWXL column (7.5 X 300 mm) and eluted with a flow rate of 0.5 ml/ min. The temperature of the sample in the sample loop, elution buffer and the column was maintained constant. (From Physiol Chem. Phys. Med. NMR, 21, 222 226 (1989)).

An alternative view is that an enzyme denatures in two stages reversible conversion of active native enzyme (N) to an inactive unfolded state (U), followed by irreversible conversion to inactivated enzyme (T) ... 

In the following experiment, we will study /3-galactosidase denaturation by guanidine chloride following OD of PNP at 405 nm. Enzyme denaturation induces a loss in its activity, which will be observed by the decrease in the OD. 

In the glucose oxidase system, dissolved oxygen concentration as well as glucose levels will influence dehvery response requiring close control of mass-transfer limitations. For both systems containing a protein component, stabihty factors may limit operational hfetime. This may be particularly severe in the case of glucose oxidase where the reaction product H2O2 will accelerate enzyme denaturation unless it is rapidly removed by diffusion or reaction with a second enzyme (peroxidase or catalase). 

For example, cholinesterase may be entrapped in a gel prepared from 5% crosslinker and 15% monomer in aqueous solution.21 Under these conditions, 56% of the total enzyme activity was retained. At higher total [monomer] + [cross-linker], the enzyme denatures, while higher [cross-linker] values yielded less entrapped enzyme. 

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